Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli

Michael A. Sinev; Elena V. Sineva; Varda Ittah; Elisha Haas

doi:10.1016/S0014-5793(96)01195-7

Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli

Michael A. Sinev, Elena V. Sineva, Varda Ittah, Elisha Haas

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Abstract

Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223-227). The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by a ~9 Å decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E. coli AK.

Original language	English
Pages (from-to)	273-276
Number of pages	4
Journal	FEBS Letters
Volume	397
Issue number	2-3
DOIs	https://doi.org/10.1016/S0014-5793(96)01195-7
State	Published - 18 Nov 1996

Keywords

Adenylate kinase
Energy transfer
Probe
Site-directed mutagenesis
Substrate inhibition
Time-resolved fluorescence

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10.1016/S0014-5793(96)01195-7

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title = "Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli",

abstract = "Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223-227). The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by a ~9 {\AA} decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E. coli AK.",

keywords = "Adenylate kinase, Energy transfer, Probe, Site-directed mutagenesis, Substrate inhibition, Time-resolved fluorescence",

author = "Sinev, {Michael A.} and Sineva, {Elena V.} and Varda Ittah and Elisha Haas",

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AU - Sinev, Michael A.

AU - Sineva, Elena V.

AU - Ittah, Varda

AU - Haas, Elisha

PY - 1996/11/18

Y1 - 1996/11/18

N2 - Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223-227). The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by a ~9 Å decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E. coli AK.

AB - Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223-227). The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by a ~9 Å decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E. coli AK.

KW - Adenylate kinase

KW - Energy transfer

KW - Probe

KW - Site-directed mutagenesis

KW - Substrate inhibition

KW - Time-resolved fluorescence

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Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli

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Keywords

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